Cecilia Poderoso

A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis.

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein –a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser232. Directed mutagenesis of Ser232 to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.

Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria

Mitochondria are major cellular sources of hydrogen peroxide (H2O2), the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H2O2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H2O2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs) orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H2O2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 µM H2O2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 µM H2O2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei), the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs) facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H2O2 level. Like this, high H2O2 or directed mutation of redox-sensitive ERK2 Cys214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to increase H2O2 yield should disrupt synchronized MAPK oxidations and the regulation of cell cycle leading cells to remain in a proliferating phenotype.

Mitochondrial Fusion Is Essential for Steroid Biosynthesis

Although the contribution of mitochondrial dynamics (a balance in fusion/fission events and changes in mitochondria subcellular distribution) to key biological process has been reported, the contribution of changes in mitochondrial fusion to achieve efficient steroid production has never been explored. The mitochondria are central during steroid synthesis and different enzymes are localized between the mitochondria and the endoplasmic reticulum to produce the final steroid hormone, thus suggesting that mitochondrial fusion might be relevant for this process. In the present study, we showed that the hormonal stimulation triggers mitochondrial fusion into tubular-shaped structures and we demonstrated that mitochondrial fusion does not only correlate-with but also is an essential step of steroid production, being both events depend on PKA activity. We also demonstrated that the hormone-stimulated relocalization of ERK1/2 in the mitochondrion, a critical step during steroidogenesis, depends on mitochondrial fusion. Additionally, we showed that the SHP2 phosphatase, which is required for full steroidogenesis, simultaneously modulates mitochondrial fusion and ERK1/2 localization in the mitochondrion. Strikingly, we found that mitofusin 2 (Mfn2) expression, a central protein for mitochondrial fusion, is upregulated immediately after hormone stimulation. Moreover, Mfn2 knockdown is sufficient to impair steroid biosynthesis. Together, our findings unveil an essential role for mitochondrial fusion during steroidogenesis. These discoveries highlight the importance of organelles’ reorganization in specialized cells, prompting the exploration of the impact that organelle dynamics has on biological processes that include, but are not limited to, steroid synthesis.

Hormonal activation of a kinase cascade localized at the mitochondria is required for StAR protein activity

It is known that ERK1/2 and MEK1/2 participate in the regulation of Star gene transcription. However, their role in StAR protein post-transcriptional regulation is not described yet. In this study we analyzed the relationship between the MAPK cascade and StAR protein phosphorylation and function. We have demonstrated that (a) steroidogenesis in MA-10 Leydig cells depends on the specific of ERK1/2 activation at the mitochondria; (b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in this organelle; (c) active ERK1/2 interacts with StAR protein, leads to StAR protein phosphorylation at Ser(232) only in the presence of cholesterol; (d) directed mutagenesis of Ser(232) (S232A) inhibited in vitro StAR protein phosphorylation by ERK1; (e) transient transfection of MA-10 cells with StAR S232A cDNA markedly reduced the yield of progesterone production. We show that StAR protein is a substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric complex that regulates cholesterol transport.

MAPK Phosphatase-1 (MKP-1) Expression Is Up-Regulated by hCG/cAMP and Modulates Steroidogenesis in MA-10 Leydig Cells

MAP kinases (MAPKs), such as ERK1/2, exert profound effects on a variety of physiological processes. In steroidogenic cells, ERK1/2 are involved in the expression and activation of steroidogenic acute regulatory protein, which plays a central role in the regulation of steroidogenesis. In MA-10 Leydig cells, LH and chorionic gonadotropin (CG) trigger transient ERK1/2 activation via protein kinase A, although the events that lead to ERK1/2 inactivation are not fully described. Here, we describe the hormonal regulation of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates MAPKs, in MA-10 cells. In our experiments, human CG (hCG)/cAMP stimulation rapidly and transiently increased MKP-1 mRNA levels by a transcriptional action. This effect was accompanied by an increase in protein levels in both nuclear and mitochondrial compartments. In cells transiently expressing flag-MKP-1 protein, hCG/cAMP promoted the accumulation of the recombinant protein in a time-dependent manner (10-fold at 1 h). Moreover, hCG/cAMP triggered ERK1/2-dependent MKP-1 phosphorylation. The blockade of cAMP-induced MAPK kinase/ERK activation abated MKP-1 phosphorylation but only partially reduced flag-MKP-1 protein accumulation. Together, these results suggest that hCG regulates MKP-1 at transcriptional and posttranslational level, protein phosphorylation being one of the mechanisms involved in this regulation. Our study also demonstrates that MKP-1 overexpression reduces the effects of cAMP on ERK1/2 phosphorylation, steroidogenic acute regulatory gene promoter activity, mRNA levels, and steroidogenesis, whereas MKP-1 down-regulation by small interfering RNA produces opposite effects. In summary, our data demonstrate that hCG regulates MKP-1 expression at multiple stages as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis.

Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR.

Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase.

The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.

An Acth-Activated Protein Tyrosine Phosphatase (PTP) is Modulated by Pka-Mediated Phosphorylation

In adrenal cortex, ACTH regulation of steroidogenesis depends on PKA-dependent serine/threonine phosphorylation and also on the activity of protein tyrosine phosphatases (PTPs). In addition, ACTH increases total PTPs involving at least three soluble PTPs (50, 82 and 115 kDa). Serine/threonine phosphorylation of these enzymes themselves could be a regulatory mechanism of their activity since the increase of total PTP activity is dependent on PKA-activation. In this report we analyzed the effect of in vitro phospho-dephosphorylation processes on the activity displayed by the ACTH-activated PTP of 115 kDa. Using an in-gel PTP assay we demonstrate that dephosphorylation catalyzed by potato acid phosphatase (PAP) reduces the activity of the 115 kDa PTP present in ZF from ACTH-treated animals and PKA-mediated phosphorylation reverses this effect.

THE OBLIGATORY ACTION OF PROTEIN TYROSINE PHOSPHATASES IN ACTH-STIMULATED STEROIDOGENESIS IS EXERTED AT THE LEVEL OF StAR PROTEIN

A key regulatory step in the steroidogenic hormones signaling pathway is the synthesis of steroidogenic acute regulatory protein (StAR). This protein facilitates the delivery of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. ACTH and LH pathway also includes tyrosine dephosphorylation processes. Indeed, our previous studies have demonstrated that both hormones increase protein tyrosine phosphatase (PTP) activity by a PKA-dependent mechanism and that the action of PTPs is required for the stimulation of steroid biosynthesis in adrenal and Leydig cells. In order to test the putative relationship between PTP activity and StAR protein induction in adrenocortical cells, in the present study we evaluated steroid production and StAR protein level in Y1 adrenocortical cells under PTP inhibition. Phenylarsine oxide (PAO), a powerful cell permeable PTP inhibitor, reduced ACTH-stimulated steroidogenesis in a concentration-dependent fashion. A concentration of 2.5 µM of this compound inhibited steroid synthesis in a 56% (ACTH = 318 ± 30, ACTH + PAO = 145 ± 18 ng progesterone/mL, P < 0.001) and also abrogated StAR protein induction. Phenylarsine oxide reduced the protein level after 60 min and this effect still remained at 120 min. A second PTP inhibitor, benzyl phosphonic acid, acting by a different mechanism, reproduced PAO effects on both steroidogenesis and StAR protein. Taken together, these results indicate that PTP activity participates in StAR protein induction and led us to attribute to the PKA-mediated PTP activation in steroidogenic systems a functional role, as mediator of StAR protein induction.

The spatial and temporal regulation of the hormonal signal. Role of mitochondria in the formation of a protein complex required for the activation of cholesterol transport and steroids synthesis.

The mitochondria are critical for steroidogenesis since the ability of cholesterol to move into mitochondria to be available for cytochrome P450, CYP11A1, determines the efficacy of steroid production. Several proteins kinases, such as PKA, MEK and ERK which are essential to complete steroidogenesis, form a mitochondria-associated complex. The protein-protein interactions between kinases and key factors during the transport of cholesterol takes place in the contact sites between the two mitochondrial membranes; however, no mitochondrial targeting sequence has been described for these kinases. Here we discuss the possibility that mitochondrial reorganization may be mediating a compartmentalized cellular response. This reorganization could allow the physical interaction between the hormone-receptor complex and the enzymatic and lipidic machinery necessary for the complete steroid synthesis and release. The movement of organelles in specialized cells could impact on biological processes that include, but are not limited to, steroid synthesis.